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Cardiovascular Diseases Poster Session






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
Board

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The prevalence and linkage disequilibrium of three methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms varies in different ethnic groups.


Contact Person: David E. C. Cole (davidec.cole@utoronto.ca)


Introduction

The MTHFR gene has been cloned and sequenced and disease-associated mutations identified (1; 2). In particular, a prevalent C->T polymorphism at nucleotide position 677 results in a conservative Ala Val (A223V) replacement characterized by reduced enzyme activity and thermolability (tMTHFR) (3). The 677T variant is significantly correlated with reduced enzyme activity and increased circulating levels of homocysteine (3), and is likely to be an important genetic factor contributing to the variation in total plasma homocysteine (tHcy), which is recognized as an independent predictor of arteriosclerotic disease, including stroke, myocardial infarction, and peripheral vascular disease (4-6). The C677T polymorphism has also been shown to be a genetic risk factor in the induction of neural tube defects (7).

Another MTHFR mutation, 1298(A->C), has been reported, which should be expressed as a substitution of glutamate by an alanine residue in the regulatory domain of the MTHFR protein (8; 9). Van der Put et al. (8) suggest that this A1298C variant results in decreased MTHFR activity, which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous 1298C genotype has been associated with higher plasma tHcy, although there appears to some interaction between the C677T and A1298C loci, at least in the population studies by van der Put et al. (8) and Weisberg et al. (9). In these studies, double heterozygosity was associated with reduced MTHFR activity, higher tHcy, and decreased plasma folate levels, which may have clinical effects similar to those observed in homozygotes for the 677T allele (8; 9).

Weisberg et al. (9) have also described a third polymorphism in the MTHFR gene [1317(T->C)]. This is a silent mutation in the third codon position, so that the phenylalanine at that position remains unchanged. The 1317C polymorphism creates a potential MboII site, and although it was first identified as a broadening of the MboII restriction digest using primers previously described (8) in genotyping the 1298 locus, not all patients with the 1317C polymorphism demonstrated the band broadening. In the survey by Weisberg et al. (9) this mutation was found to have an allele frequency of 5% in their controls, but the prevalence rose to 39% in an independent ethnospecific sample of persons of African ansestry.

Because of the increasing utilization of the C677T allele as an indicator of predisposition for hyperhomocysteinemia and its associated consequences, we considered it important to determine the prevalence of these alleles and their linkage disequilibrium in the major sub-populations of different ethnic backgrounds in North America.

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Materials and Methods

Our sample sets were derived from North American urban centres. Both caucasian (n=197) and asian (n=51) sample sets were derived from healthy adults in the Toronto area and ethnicity was established by questionnaire. Although those reporting mixed ethnic backgrounds were excluded, geographic origins or genetic admixture were not identified and the self-reported ethnic background was used through out. Similar criteria applied to the afroamerican sample (n=51) recruited from the New York city conurbation.

The C677T genotype was analysed by PCR using primers [TGAAGGAGAA GGTGTCTGCG GGA (forward) and AGGACGGTGC GGTGAGAGTG' (reverse)], followed by and restriction digestion with the Hinf1 enzyme (3). The 677T polymorphism results in a 175 bp product, separated by agarose gel electrophoresis, and detected by ethidium bromide staining.

The A1298C locus was genotyped by mutation-selective PCR (MS-PCR) using two forward primers (CAAGGAGGAG CTGCTGAAGA TGTGGGGCCA GGAGCTGACC AGTGTAGA for the detection of the 1298A , and GGAGGAGCTG ACCAGTGATG C for the detection of 1298C) and a single reverse primer (GACCCAGCCT GTCTTTGCCT). MS-PCR generates a 302bp product for the 1298A allele and a 275bp product for the 1298C. If the PCR products are digested with BbsI , the T1317C polymorphism can be distinguished. The BbsI enzyme cleaves the 1317C PCR product, producing a 247bp fragment. This technique also allows for phase determination orientation of 1317 relative to 1298.

Frequencies were calculated by counting chromosomal alleles from the observed genotypes. Differences between populations were tested for heterogeneity using the chi-square analysis in Instat (GraphPad Software Inc.) with statistical significance at the 5% level.

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Results

In all 3 ethnic groups combined, the 677T variant was relatively common (176/596 alleles, 30%). As expected, it was least frequent in the afro-american sample set (13/102 alleles, 12.7%). This frequency was significantly less than in the North American asians (30/100,30%, p=0.01) or caucasians (133/394,33.7%, p =0.001).

A similar pattern emerges for the A1298C polymorphism. The 1298C allele was significantly lower (p=0.01) in afro-americans (16/102, 15.7%), compared to caucasians (118/394,29.9%), but the frequency in North-American asians (20/100, 20%) does not reach statistical significance. On the other hand, the 1317C polymorphism was exceedingly rare in non-african groups. We observed only this variant in only 1 of 494 caucasians and asian chromosomes. In the afro-american sample set, the frequency was 37.3% (38/102 alleles), which was clearly different from the other groups.

When probed in a preliminary way by chi-square tests, the 3 3 genotype matrices showed evidence of linkage disequilibrium. For the composite 677+1298 genotypes in caucasians, this disequilibrium was substantial (chi-square = 24.7, p < 0.001). An individual with at least one copy of the 677T variant had a 42% chance of bearing at least one 1298C variant. Comparable data in the smaller asian subset showed a similar correlation (chi-square = 7.44, p=0.12).

For the afro-american population, it was possible to analyse three two-way sets. There was no disequilibrium between the 677+1298 (chi-square = 1.35, p=0.85), nor the african-specific 1317C variant and the other two sites (chi-square = 4.3, p=0.37 for the 677 polymorphism and chi-square = 6.69, p=0.15).

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Discussion and Conclusion

Our study was designed to determine if preliminary data on additional MTHFR polymorphisms described in the Dutch (8)and French-Canadian (9) populations could be extended to the 3 major ethnic groups that remain self-identified in large, multiracial North American cities. Our data showed heterogeneity in the prevalence of the T677 allele among the ethnic groups studied, which is in keeping with other studies (9-12). With the allele frequency in persons of african descent (13%, p<0.001) as statistically lower than caucasians (34%). There was also a statistical difference in allele frequency in our asian subset (30%, p<0.01) compared to caucasians, but we recognize that there appears to be a wide variation in the allele frequencies from those asian sample sets derived from specific geographic and ethnoracially distinguishable sub-populations (13).

We could find no other reports on the frequency of the A1298C polymorphism in relation to ethnic origin. Our sample sets suggest that there is a statistically lower frequency of the 1298C allele in afro-americans (15%, p<0.01) compared to caucasians (30%). Although there was not significant difference in the asian group, the sample size was small, and further studies are indicated.

The third variant, T1317C, was found in more than a third of the chromosomes studies in our afro-american subset, in keeping with the previous report indicating an allele frequency of 39% (7/18 chromosomes) (9). With the methodology for the detection of A1298C and T1317C outlined in this report it is possible to determine the orientation of the polymorphic sites. In the African population studied, the 8 subjects who were doubly heterozygous for both 1317C and 1298C, all had the mutations in trans, suggesting additional linkage disequilibrium. Whether this site has any functional significance remains obscure.

Because research studies have successfully used single functional polymorphisms to draw powerful conclusions about genetic predisposition to disease, it is often expected that single locus genotyping should be transferrable to clinical practice, as long as appropriate interpretive counselling is offered. While there is no doubt that the 677T genotype could be clinically relevant, the possibility that other polymorphisms, such as 1298C, also contribute should give pause to those who would promote the rapid transfer of genotyping to the routine setting. In this context, genetic studies of the paraoxonase locus (PON1) are relevant (14). Initially, the associative analysis was conducted on the one known polymorphic site, the Q191K polymorphism and a strong effect was found with coronary artery disease in type II diabetes mellitus (15). Then, attention was drawn to the fact that a second site, M54L, also showed strong association with cardiovascular disease (16), and the exact relationship between these two loci and long-term outcomes is still being established (17).

Both 677T and 1298C polymorphisms should result in amino acid substitutions and functional alterations in the MTHFR protein and may be independently associated with a significantly elevated tHcy phenotype (9). However, the variation in linkage disequilibrium we observe will be a significant confounding factor in interpretation of a single genotype. Similar disequilibrium between 677T and 1298C (chi-square=55.6, p<0.0001) was observed in caucasian parents and children with neural tube defects by Weisberg et al. (9), but it is possible that this confounding will be ethno-specific.

In summary, our data indicate that the assumption that genotyping either C677T or A1298C locus alone is sufficient to identify predisposition to hyperhomocystinemia may be violated to differing degrees, depending on the linkage disequilibrium in the population studied, and the degree to which tHcy is modified by other factors, such as folate status. Further studies of this complex interaction are warranted, if the relationship of MTHFR genotype to risks for arteriosclerotic disease is to be fully understood.

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References

1. Goyette P, Sumner JS, Milos R, Duncan AM, Rosenblatt DS, Matthews RGRR. Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification Nature Genet. 1994;7:195-200.

2. Rozen R. Molecular genetic aspects of hyperhomocysteinemia and its relation to folic acid. Clin.Invest.Med. 1996;19:171-178.

3.Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk fsctor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nature Genet. 1995;10:111-113.

4. Miner SES, Evrovski J, Cole DEC. Clinical chemistry and molecular biology of homocysteine metabolism: An update. Clin.Biochem. 1997;30:189-201.

5. Boushey CJ, Beresford SA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 1995;274:1049-1057.

6. Stein JH, McBride PE. Hyperhomocysteinemia and atherosclerotic vascular disease: pathophysiology, screening, and treatment. off. Arch.Intern.Med 1998;158:1301-1306.

7. van der Put NM, Eskes TK, Blom HJ. Is the common 677C-->T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects? A meta-analysis. QJM. 1997;90:111-115.

8. van der Put NM, Gabreels F, Stevens EM, et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am.J.Hum.Genet. 1998;62:1044-1051.

9. Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol.Genet.Metab. 1998;64:169-172.

10. Franco RF, Araujo AG, Guerreiro JF, Elion J, Zago MA. Analysis of the 677 C-->T mutation of the methylenetetrahydrofolate reductase gene in different ethnic groups. Thromb.Haemost. 1998;79:119-121.

11. Sacchi E, Tagliabue L, Duca F, Mannucci PM, Bianchi A. High frequency of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in northern Italy. Thrombosis and Haemostasis 1997;78:963-964.

12. Pepe G, Camacho VO, Giusti B, et al. Heterogeneity in world distribution of the thermolabile C677T mutation in 5,10-methylenetetrahydrofolate reductase. Am.J.Hum.Genet. 1998;63:917-920.

13. Gregg J, Bando JM, Crandall BF, Grody WW. Frequencies of two common polymorphisms in the methylenetetrahydrofolate reductase gene in four different ethnic American populations. Am.J.Hum.Genet. 1998;63 Suppl 1:A213

14. LaDu BN. Structural and functional diversity of paraoxonases. Nature Medicine 1996;2:1186-1187. 15. Ruiz J, Blanche H, James RW, et al. Gln-Arg192 polymorphism of paraoxonase and coronary heart disease in type 2 diabetes. Lancet 1995;346:869-872.

16. Garin M-CB, James RW, Dussoix P, et al. Paraoxonase polymorphism met-leu54 is associated with modified serum concentrations of the enzyme. J.Clin.Invest. 1997;99:62-66.

17. Sanghera DK, Aston CE, Saha N, Kamboh MI. DNA polymorphisms in two paraoxonase genes (PON1 and PON2) are associated with the risk of coronary heart disease. Am.J.Hum.Genet. 1998;62:36-44.

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Langman, L.J.; Wong, B.Y.-L.; Boggis, C.; Rubin, L.A.; Cole, D.E.C.; (1998). The prevalence and linkage disequilibrium of three methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms varies in different ethnic groups.. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/cvdisease/langman0264/index.html
© 1998 Author(s) Hold Copyright